Milo: liver cirrhosis analysis

Introduction

In this notebook we demonstrate how to use Milo to detect abherrant cell states in diseased tissues, using a dataset of hepatic non-parenchymal cells isolated from 5 healthy and 5 cirrhotic human livers. Ramachandran et al. 2019 (GEO accessiion: GSE136103).

Installation

Added by DRT 2021-12-03

Updated by DRT 2023-11-29

source("getReqdPkgs.r")

pkgs <- c("SingleCellExperiment","scater","scran","Seurat","miloR",
  "tidyverse","patchwork","igraph","ggplot2",
  "ggrastr","msigdbr","clusterProfiler",
  "RColorBrewer","cowplot","devtools")

extra_pkgs <- c("irlba","Matrix") # NOTE these must be installed from source to make code work; see next code block

invisible(getReqdPkgs(pkgs))

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")' for
details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
All required CRAN and BioC packages have previously been installed

NOTE

As of 2023-11-29, the runPCA function in scater breaks due to change in Matrix library; must downgrade to Matrix 1.6.1 Downloaded v 1.6.1 from https://cran.r-project.org/src/contrib/Archive/Matrix/

installing from source (compiling on your computer, rather than installing from a binary) may also be needed. On a Mac, this requires gfortran to be installed. See here and link to download gfortran 12.2

See this link: https://github.com/bwlewis/irlba/issues/70

# install.packages("irlba", type = "source")
# install.packages("Matrix", type = "source") # , version="1.6.1"

library(scater)

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Welcome to Bioconductor

    Vignettes contain introductory material; view with 'browseVignettes()'. To cite Bioconductor, see
    'citation("Biobase")', and for packages 'citation("pkgname")'.


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library(scran)
library(miloR)

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library(tidyverse)

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library(patchwork)
library(igraph)

Attaching package: ‘igraph’

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library(ggplot2)
library(ggrastr)
library(msigdbr)
library(cowplot)

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library(Matrix)

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library(irlba)
library(RColorBrewer)
library(SingleCellExperiment)

Define some global variables

Location to save output files and whether to overwrite output files.

OUTPUT_DIR <- "~/dropbox-vu/temp/milo_output"
OVERWRITE <- FALSE

Load data

We downloaded the dataset and annotations stored in Seurat object from here, as indicated by the authors. The files are on DropBox and can be accessed directly with the code below.

# load("/nfs/team205/ed6/data/Ramachandran2019_liver/tissue.rdata")
load(url("https://www.dropbox.com/s/bq816h74gmh84gu/tissue.rdata?dl=1"))

Loading required package: Seurat
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Attaching package: ‘sp’

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## Convert to SingleCellExperiment
liver_sce <- SingleCellExperiment(assay = list(counts=tissue@raw.data, logcounts=tissue@data),
                                  colData = tissue@meta.data)

liver_sce

class: SingleCellExperiment 
dim: 23498 58358 
metadata(0):
assays(2): counts logcounts
rownames(23498): FO538757.2 AP006222.2 ... CTA-126B4.7 LINC01423
rowData names(0):
colnames(58358): Healthy1_Cd45+_AAACCTGCAGTATCTG Healthy1_Cd45+_AACTGGTTCATGGTCA ...
  Cirrhotic3_Cd45-_TTTGTCATCCAGGGCT Cirrhotic3_Cd45-_TCTGGAAGTCATCCCT
colData names(10): nGene nUMI ... annotation_indepth annotation_lineage
reducedDimNames(0):
mainExpName: NULL
altExpNames(0):

Preprocessing

We use the same number of highly variable genes and principal components used by the authors of the original study.

Feature selection

Select highly variable genes

dec_liver <- modelGeneVar(liver_sce)

fit_liver <- metadata(dec_liver)
plot(fit_liver$mean, fit_liver$var, xlab="Mean of log-expression",
    ylab="Variance of log-expression")

hvgs <- getTopHVGs(dec_liver, n=3000)

Dimensionality reduction

set.seed(42)
liver_sce <- runPCA(liver_sce, subset_row=hvgs, ncomponents=11)
liver_sce <- runUMAP(liver_sce, dimred="PCA", ncomponents=2)

Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’

scater::plotUMAP(liver_sce, colour_by="condition", point_alpha=1,  point_size=0.5)

scater::plotUMAP(liver_sce, colour_by="dataset", point_alpha=0.3,  point_size=0.5)

scater::plotUMAP(liver_sce, colour_by="annotation_lineage", point_alpha=0.3,  point_size=0.5, text_by='annotation_lineage')

Notably, this dataset doesn’t appear to display a batch effect

Note: This step is not required. It generates a large file (even larger than raw data) and then reloads it.

# saveRDS(liver_sce, "~/dropbox-vu/temp/milo_data/Ramachandran2019_liver/liver_SCE_20210225.RDS")
# liver_sce <- readRDS(url("https://www.dropbox.com/s/z0aopf616b1urvj/liver_SCE_20210225.RDS?dl=1"))

Differential Abundance (DA) analysis with Milo

We test for differential abundance between healthy and cirrhotic livers. We start by defining neighbourhoods with refined sampling on the KNN graph. We inspect the size of neighbourhoods.

liver_milo <- Milo(liver_sce)

## Build KNN graph
liver_milo <- buildGraph(liver_milo, d = 11, k=30)

Constructing kNN graph with k:30

## Compute neighbourhoods with refined sampling
liver_milo <- makeNhoods(liver_milo, k=30, d=11, prop = 0.05, refined=TRUE)

Checking valid object
Running refined sampling with reduced_dim

plotNhoodSizeHist(liver_milo, bins=150)

Then we make a design matrix for the differential test, assigning samples to biological conditions.

colData(liver_milo)[['sort']] <- str_remove(colData(liver_milo)[['dataset']], ".+_")
colData(liver_milo)[['sort']] <- str_remove(colData(liver_milo)[['sort']], "A|B")

liver_meta <- as_tibble(colData(liver_milo)[,c("dataset","condition", 'sort')])
liver_meta <- distinct(liver_meta) %>%
  mutate(condition=factor(condition, levels=c("Uninjured", "Cirrhotic"))) %>%
  column_to_rownames("dataset")

Now we can count cells in neighbourhoods and perform the DA test.

NOTE: Executing calcNhoodDistance() function takes a long time (>30 min)

liver_milo <- countCells(liver_milo, samples = "dataset", meta.data = data.frame(colData(liver_milo)[,c("dataset","condition",'sort')]) )

Checking meta.data validity
Counting cells in neighbourhoods

liver_milo <- calcNhoodDistance(liver_milo, d=11)

'as(<dgTMatrix>, "dgCMatrix")' is deprecated.
Use 'as(., "CsparseMatrix")' instead.
See help("Deprecated") and help("Matrix-deprecated").

milo_res <- testNhoods(liver_milo, design = ~ condition, design.df = liver_meta[colnames(nhoodCounts(liver_milo)),])

Using TMM normalisation
Performing spatial FDR correction withk-distance weighting

milo_res_sort <- testNhoods(liver_milo, design = ~ sort + condition, design.df = 
                                liver_meta[colnames(nhoodCounts(liver_milo)),])

Using TMM normalisation
Performing spatial FDR correction withk-distance weighting

compare_da_df <- left_join(milo_res_sort, milo_res, by="Nhood", suffix=c("_sort", "_nosort")) %>%
  {annotateNhoods(liver_milo, ., 'annotation_lineage')} 

compare_da_df %>%
  ggplot(aes(-log10(SpatialFDR_sort), -log10(SpatialFDR_nosort))) +
  geom_point(size=0.8) +
  geom_point(data=. %>% filter(annotation_lineage=="Endothelia"), color="red")

plot(milo_res_sort$SpatialFDR, milo_res$SpatialFDR)

Exploration of Milo DA results

We can start by looking at some basic stats

pval_hist <- milo_res %>%
  ggplot(aes(PValue)) +
  geom_histogram(bins=50) +
  theme_bw(base_size=14)

volcano <-
  milo_res %>%
  ggplot(aes(logFC, -log10(SpatialFDR))) +
  geom_point(size=0.4, alpha=0.2) +
  geom_hline(yintercept = -log10(0.1)) +
  xlab("log-Fold Change") +
  theme_bw(base_size=14)

pval_hist + volcano

The distribution of P-values looks sensible and from the volcano plot we can see that we have identified some DA neighbourhoods at 10% FDR.

We can visualize DA neighbourhoods building an abstracted graph.

NOTE: This takes a while to process! Can take >30 min.

liver_milo <- buildNhoodGraph(liver_milo)
plotNhoodGraphDA(liver_milo, milo_res, alpha = 0.1, size_range=c(2,6))

Note: This step is not required. It generates a large file (even larger than raw data) and then reloads it.

## Save milo object and results
# saveRDS(liver_milo,"~/dropbox-vu/temp/milo_data/Ramachandran2019_liver/liver_milo_20210225.RDS")
# write_csv(milo_res,"~/dropbox-vu/temp/milo_data/Ramachandran2019_liver/liver_results_20210225.csv")

# liver_milo <- readRDS("~/liver_milo_20201008.RDS")
liver_milo <- readRDS(url("https://www.dropbox.com/s/xdp07789c5hoen3/liver_milo_20210225.RDS?dl=1"))
# milo_res <- read_csv("/nfs/team205/ed6/data/Ramachandran2019_liver/liver_results_20201008.csv")
milo_res <- read_csv(url("https://www.dropbox.com/s/i1l3aep1py5wirf/liver_results_20210225.csv?dl=1"))

Making figures for the manuscript

colourCount = length(unique(liver_milo$annotation_lineage))
getPalette = colorRampPalette(brewer.pal(9, "Set2"))

Warning: n too large, allowed maximum for palette Set2 is 8
Returning the palette you asked for with that many colors

umap_df <- data.frame(reducedDim(liver_milo, "UMAP"))
colnames(umap_df) <- c("UMAP_1", "UMAP_2")

umap1 <- cbind(umap_df, annotation_lineage=liver_milo$annotation_lineage) %>%
  ggplot(aes(UMAP_1, UMAP_2, color=as.character(annotation_lineage))) +
  geom_point_rast(size=0.1, alpha=0.5, raster.dpi = 800) +
  ggrepel::geom_text_repel(data = . %>%
              group_by(annotation_lineage) %>%
              summarise(UMAP_1=mean(UMAP_1), UMAP_2=mean(UMAP_2)),
            aes(label=annotation_lineage), color="black", size=6
            ) +
  scale_color_manual(values=getPalette(colourCount)) +
  guides(color="none") +
  xlab("UMAP1") + ylab("UMAP2") +
  coord_fixed() +
  theme_classic(base_size = 22) +
  theme(axis.text = element_blank(), axis.ticks = element_blank())

fn_liver_umap1 <- file.path(OUTPUT_DIR,"/liver_v2/liver_umap1_new.pdf")

if(OVERWRITE | !file.exists(fn_liver_umap1)) {
  ggsave(fn_liver_umap1, height = 7, width = 8)
} else {
  message(cat(fn_liver_umap1,"exists and will not be overwritten\n"))
}

~/dropbox-vu/temp/milo_output//liver_v2/liver_umap1_new.pdf exists and will not be overwritten

umap2 <-
  cbind(umap_df, condition=as.character(liver_milo$condition)) %>%
  ggplot(aes(UMAP_1, UMAP_2, color=condition)) +
  geom_point_rast(size=0.1, alpha=0.5, raster.dpi = 800) +
  scale_color_brewer(palette="Set1", name='') +
  xlab("UMAP1") + ylab("UMAP2") +
  coord_fixed() +
  guides(color='none') +
  facet_wrap(condition~., ncol=1) +
  theme_nothing(font_size = 22) +
  theme(axis.text = element_blank(), axis.ticks = element_blank(), legend.position=c(0.9,0.9),
        strip.background = element_rect(color=NA), strip.text = element_text(size=22))


fn_liver_umap2 <- file.path(OUTPUT_DIR,"/liver_v2/liver_umap2_new.pdf")

if(OVERWRITE | !file.exists(fn_liver_umap1)) {
  ggsave(fn_liver_umap2, height = 7, width = 8)
} else {
  message(cat(fn_liver_umap2,"exists and will not be overwritten\n"))
}

~/dropbox-vu/temp/milo_output//liver_v2/liver_umap2_new.pdf exists and will not be overwritten

nh_graph_pl <- plotNhoodGraphDA(liver_milo, milo_res, alpha = 0.1, size_range=c(1,4)) +
  theme(legend.text = element_text(size=20), legend.title = element_text(size=22)) +
  coord_fixed()

fn_nh_graph <- file.path(OUTPUT_DIR,"/liver_v2/liver_graph_new.pdf")

if(OVERWRITE | !file.exists(fn_nh_graph)) {
  ggsave(fn_nh_graph, height = 7, width = 8)
} else {
  message(cat(fn_nh_graph,"exists and will not be overwritten\n"))
}

~/dropbox-vu/temp/milo_output//liver_v2/liver_graph_new.pdf exists and will not be overwritten

fig4_top <- (umap1 | umap2 | nh_graph_pl) +
  plot_layout(widths = c(3,1,3))

fig4_top

Explore DA neighbourhoods by cell type

Next, we can check the cell types where we observe most differences between healthy and cirrhotic cells, by taking the most frequent cell type in each neighbourhood.

milo_res <- milo_res[,!str_detect(colnames(milo_res), "annotation_lineage")]

# Add annotation of most frequent cell type per nhood to milo results table
milo_res <- annotateNhoods(liver_milo, milo_res, "annotation_indepth")
anno_df <- data.frame(liver_milo@colData) %>%
  distinct(annotation_lineage, annotation_indepth)
milo_res <- left_join(milo_res, anno_df, by="annotation_indepth")

We first check that neighbourhoods are sufficiently homogeneous

frac_hist <- ggplot(milo_res, aes(annotation_indepth_fraction)) +
  geom_histogram(bins=30) +
  xlab("Fraction of cells in \nmost abundant cluster") +
  ylab("# neighbourhoods") +
  theme_bw(base_size=14)

frac_hist

Filter nhoods with homogeneous composition

milo_res$annotation_indepth[milo_res$annotation_indepth_fraction < 0.6] <- NA
milo_res$annotation_lineage[milo_res$annotation_indepth_fraction < 0.6] <- NA

We can recover all the clusters where DA was detected in the original paper

group.by = "annotation_indepth"
paper_DA <- list(cirrhotic=c("MPs (4)","MPs (5)",
                             "Endothelia (6)", "Endothelia (7)",
                             "Mes (3)",
                             "Tcells (2)",
                             "Myofibroblasts"
                             ),
                 healthy=c("MPs (7)",
                           "Endothelia (1)",
                           "Tcells (1)", "Tcells (3)","Tcells (1)",
                           "ILCs (1)"
                           )
                 )

expDA_df <- bind_rows(
  data.frame(annotation_indepth = paper_DA[["cirrhotic"]], pred_DA="cirrhotic"),
  data.frame(annotation_indepth = paper_DA[["healthy"]], pred_DA="healthy")
  )

pl1 <- milo_res %>%
  left_join(expDA_df) %>%
  mutate(is_signif = ifelse(SpatialFDR < 0.1, 1, 0)) %>%
  mutate(logFC_color = ifelse(is_signif==1, logFC, NA)) %>%
  arrange(annotation_lineage) %>%
  mutate(Nhood=factor(Nhood, levels=unique(Nhood))) %>%
  filter(!is.na(annotation_lineage)) %>%
  ggplot(aes(annotation_indepth, logFC, color=logFC_color)) +
  scale_color_gradient2() +
  guides(color="none") +
  xlab(group.by) + ylab("Log Fold Change") +
  ggbeeswarm::geom_quasirandom(alpha=1) +
  coord_flip() +
  facet_grid(annotation_lineage~., scales="free", space="free") +
  theme_bw(base_size=22) +
  theme(strip.text.y =  element_text(angle=0),
        axis.title.y = element_blank(), axis.text.y = element_blank(), axis.ticks.y = element_blank(),
        )

pl2 <- milo_res %>%
  left_join(expDA_df) %>%
  # dplyr::filter(!is.na(pred_DA)) %>%
  group_by(annotation_indepth) %>%
  summarise(pred_DA=dplyr::first(pred_DA), annotation_lineage=dplyr::first(annotation_lineage)) %>%
  mutate(end=ifelse(pred_DA=="healthy", 0, 1),
         start=ifelse(pred_DA=="healthy", 1, 0)) %>%
  filter(!is.na(annotation_lineage)) %>%
  ggplot(aes(annotation_indepth, start, xend = annotation_indepth, yend = end, color=pred_DA)) +
  geom_segment(size=1,arrow=arrow(length = unit(0.1, "npc"), type="closed")) +
  coord_flip() +
  xlab("annotation") +
  facet_grid(annotation_lineage~.,
    # annotation_lineage~"Ramachandran et al.\nDA predictions",
             scales="free", space="free") +
  # guides(color="none") +
  scale_color_brewer(palette="Set1", direction = -1,
                     labels=c("enriched in cirrhotic", "enriched in healthy"),
                     na.translate = F,
                     name="Ramachandran et al.\nDA predictions") +
  guides(color=guide_legend(ncol = 1)) +
  theme_bw(base_size=22) +
  ylim(-0.1,1.1) +
  theme(strip.text.y = element_blank(),strip.text.x = element_text(angle=90),
        plot.margin = unit(c(0,0,0,0), "cm"), panel.grid = element_blank(),
        axis.title.x = element_blank(), axis.text.x = element_blank(), axis.ticks.x = element_blank(),
        legend.position = "bottom")

fig4_bleft <- (pl2 + pl1 +
  plot_layout(widths=c(1,10), guides = "collect") & theme(legend.position = 'top', legend.justification = 0))

fn_liver_DAcomp <- file.path(OUTPUT_DIR,"/liver_v2/liver_DAcomparison.pdf")

if(OVERWRITE | !file.exists(fn_liver_DAcomp)) {
  ggsave(fn_liver_DAcomp, height = 13, width = 8)
} else {
  message(cat(fn_liver_DAcomp,"exists and will not be overwritten\n"))
}

~/dropbox-vu/temp/milo_output//liver_v2/liver_DAcomparison.pdf exists and will not be overwritten

# ggsave("~/mount/gdrive/milo/Figures/liver_v2/liver_DAcomparison.pdf", width=8, height = 13)
# ggsave("~/dropbox-vu/temp/milo_output/liver_v2/liver_DAcomparison.pdf", width=8, height = 13)

fig4_bleft

Close-up on Endothelial lineage

endo_milo <- scater::runUMAP(liver_milo[,liver_milo$annotation_lineage=="Endothelia"],  dimred='PCA')

Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’

plotUMAP(endo_milo, colour_by = "annotation_indepth")

umap_df <- data.frame(reducedDim(endo_milo, "UMAP"))
colnames(umap_df) <- c("UMAP_1", "UMAP_2")

endo_umap <- cbind(umap_df, condition=endo_milo$condition) %>%
   ggplot(aes(UMAP_1, UMAP_2, color=condition)) +
  geom_point(size=0.3, alpha=0.5) +
  scale_color_brewer(palette="Set1", name='') +
  xlab("UMAP1") + ylab("UMAP2") +
  coord_fixed() +
  guides(color="none") +
  facet_wrap(condition~., ncol=1) +
  theme_nothing() +
  theme(axis.text = element_blank(), axis.ticks = element_blank(), legend.position=c(0.9,0.9),
        strip.background = element_rect(color=NA), strip.text = element_text(size=22))

liver_milo2 <- liver_milo
subset.nhoods <- str_detect(milo_res$annotation_indepth, "Endo")
reducedDim(liver_milo2, "UMAP")[colnames(endo_milo),] <- reducedDim(endo_milo, "UMAP") 

endo_gr <-
  plotNhoodGraphDA(
  liver_milo2, milo_res,
  subset.nhoods = which(milo_res$annotation_lineage == "Endothelia"), 
  size_range=c(1,4),
  # ) =)[1:(length()-1)], 
  alpha = 0.1
  )  +
   theme(legend.text = element_text(size=20), legend.title = element_text(size=22))
  
fig4_bright1 <- ((endo_umap + endo_gr ) + 
  plot_layout(widths = c(1,2), 
                guides = "collect"
                )) 
fig4_bright1

Can’t get code below to run

Unlcear what the problem is; data structure?

# # nh_graph <- nhoodGraph(liver_milo)[subset.nhoods,subset.nhoods]
# 
# nh_graph <- nhoodGraph(liver_milo)[na.omit(subset.nhoods)]
# 
# nh_graph <- graph_from_adjacency_matrix(nh_graph)
# 
# col_vals <- colData(liver_milo)[as.numeric(vertex_attr(nh_graph)$name), colour_by]
# V(nh_graph)$colour_by <- ifelse(milo_res[subset.nhoods,"SpatialFDR"] > 0.1, 0, milo_res[subset.nhoods,"logFC"])
# ggraph(simplify(nh_graph)) +
#       geom_edge_link0(edge_colour = "grey66", edge_alpha=0.2)   +
#       geom_node_point(aes(fill = colour_by), shape=21, size=2) +
#   scale_fill_gradient2()

Close-up on Cholangiocytes

chol_milo <- scater::runUMAP(liver_milo[,liver_milo$annotation_lineage=="Cholangiocytes"],  dimred='PCA')

Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’

plotUMAP(chol_milo, colour_by = "annotation_indepth")

plotUMAP(chol_milo, colour_by = "percent.mito")

Filter out cells that show contamination from immune cells (expression of immune markers)

keep <- logcounts(chol_milo)["CD19",] == 0 | logcounts(chol_milo)["MS4A1",] == 0
chol_milo <- chol_milo[,keep]
chol_milo <- scater::runUMAP(chol_milo,  dimred='PCA')

Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’
Found more than one class "dist" in cache; using the first, from namespace 'BiocGenerics'
Also defined by ‘spam’

plotUMAP(chol_milo, colour_by = "annotation_indepth")

umap_df <- data.frame(reducedDim(chol_milo, "UMAP"))
colnames(umap_df) <- c("UMAP_1", "UMAP_2")

chol_umap <- cbind(umap_df, condition=chol_milo$condition) %>%
   ggplot(aes(UMAP_1, UMAP_2, color=condition)) +
  geom_point(size=0.3, alpha=0.5) +
  scale_color_brewer(palette="Set1", name='') +
  xlab("UMAP1") + ylab("UMAP2") +
  coord_fixed() +
  guides(color="none") +
  facet_wrap(condition~., ncol=1) +
  theme_nothing() +
  theme(axis.text = element_blank(), axis.ticks = element_blank(), legend.position=c(0.9,0.9),
        strip.background = element_rect(color=NA), strip.text = element_text(size=22))

chol_umap

liver_milo2 <- liver_milo
subset.nhoods <- milo_res$annotation_lineage=="Cholangiocytes"
reducedDim(liver_milo2, "UMAP")[colnames(chol_milo),] <- reducedDim(chol_milo, "UMAP") 

chol_gr <-
  plotNhoodGraphDA(
  liver_milo2, milo_res,
  subset.nhoods = subset.nhoods,
  size_range=c(2,5),
  # ) =)[1:(length()-1)], 
  alpha = 0.1
  )  +
   theme(legend.text = element_text(size=22), legend.title = element_text(size=24))
  
(chol_umap + chol_gr ) + 
  plot_layout(widths = c(1,2), 
                guides = "collect"
                )

# fig4_bright1 +
#   ggsave("~/milo_output/liver_endoGraph.pdf", width=9, height = 5)  

# ggsave("~/dropbox-vu/temp/milo_output/liver_endoGraph.pdf", width=9, height = 5)  

Differential Gene Expression analysis

In a subset of lineages, we want to test for differential expression between neighbourhoods enriched in cirrhotic cells and neighbourhoods enriched.

Add nhood expression to speed-up plotting of heatmaps

liver_milo <- calcNhoodExpression(liver_milo, assay = "logcounts", subset.row = hvgs)

# DRT: stopping run-through here

Should be able to run everything above.

Endothelia

Rebuttal figure showcasing grouping

set.seed(42)
milo_res_endogroups <- groupNhoods(liver_milo, milo_res, max.lfc.delta = 2, overlap = 1)

Found 1385 DA neighbourhoods at FDR 10%
nhoodAdjacency found - using for nhood grouping

p1 <- plotNhoodGroups(liver_milo, milo_res_endogroups, 
                size_range=c(1,3)) 

milo_res_endogroups <- annotateNhoods(liver_milo, milo_res_endogroups, 'annotation_lineage')

p2 <- plotDAbeeswarm(milo_res_endogroups, group.by = 'NhoodGroup') +
  facet_grid(annotation_lineage~., scales="free", space="free")

Converting group_by to factor...

## Plot expression in T cell neighbourhoods
# markers_df <- read_csv("~/mount/gdrive/milo/STable3_Ramachandran.csv")
tcell_marker_genes <- 
  markers_df %>%
  filter(cluster %in% c("Tcell", "ILC")) %>%
  top_n(30, myAUC) %>%
  pull(gene)

Error: object 'markers_df' not found

Group endothelial cells by logFC and DA results

Calculate marker genes between the two groups

Visualize as volcano

Visualize as heatmap

(gene expression values are scaled between 0 and 1 for each gene)

GO term analysis

Cholangiocytes

Calculate marker genes between the two groups

Visualize as volcano

GO term analysis

---

Assemble figure

Assemble supplementary figure